1. Field Of The Invention
The invention relates to a method and apparatus for the determination of sugars in fluid samples. More particularly, the present invention relates to the analysis of glucose based sugars (disaccharides, oligosaccharides, and polysaccharides) in biological fluids such as blood and urine wherein free glucose may be present. Glucose is typically present in large amounts in biological fluids and may substantially interfere with the quantification of sample components which are present in smaller quantities, such as, for example, maltose, maltotriose, oligosaccharides and the like.
Presently, there is a need in clinical chemistry for an instrument and analytical technique which is capable of monitoring biological fluids for the total of the sugars present which are present in varying concentrations and when the samples to be determined are known to contain glucose and contaminating amounts. Recently, a great deal of attention has been directed towards the development of analytical methods for maltose in particular, based on the enzymatic conversions of sugars in the presence of an enzyme which is capable of reacting with the sugars to form glucose, which is a detectable end product. The present invention successfully overcomes difficulties previously encountered in sugar determination methods over wide ranges of total sugar and contaminating glucose concentrations.
2. Description Of The Prior Art
The prior art discloses that under the influence of the proper enzyme or enzymes, various sugar forms (such as disaccharides and polysaccharides) may be converted to other sugar forms. For example, U.S. Pat. No. 3,879,263 shows that in an alpha-amylase analysis, the amylase reacts with various carbohydrates to produce maltose. The maltose thus produced will react with glucoamylase, to produce glucose. These two reactions are key steps in the internal digestion of carbohydrates, especially starches.
Also, methods are known to detect various sugars in aqueous solution. U.S. Pat. No. 3,902,970 shows a method to determine glucose concentrations in solution using an amperometric detection means. The system uses the enzyme glucose oxidase to convert glucose to gluconic acid and hydrogen peroxide, with the peroxide measured in a flow-through amperometric cell to give an indication of the initial glucose concentration.
U.S. Pat. No. 4,018,651 discloses that glucose concentrations may be monitored by the rate of oxygen consumption in a glucose oxidase catalyzed reaction which consumes oxygen and produces hydrogen peroxide and gluconic acid.
Similarly, Marshall et al, "Enzymatic Determination of Maltose by Amperometric Measurement of the Rate of Oxygen Depletion", in Analyst, June 1977, Vol. 102, pp. 424-428, disclose a method for the enzymatic determination of maltose by oxygen depletion. The method converts maltose to glucose, and glucose to hydrogen peroxide and gluconic acid. The oxygen consumed in the conversion reaction is monitored by an oxygen electrode and is a measure of the glucose, and thus the maltose. The method leaves residual hydrogen peroxide which may degrade other sample components if the sample is to be used in determining multiple components. Also, the lack of the use of immobilized enzymes which can be used repeatedly, increases the cost of using the method. The contaminating glucose is removed by solution phase incubation with glucose oxidase. This also generates free hydrogen peroxide in the sample, and requires the separation of the glucose oxidase if a second substrate needs to be monitored. The method is also subject to interference by starch and sucrose.